3D printed microtissue cassettes enabling high throughput proton radiobiological assays

Background: The concept of Relative Biological Effectiveness (RBE) enables to translate the clinical experience for photon treatments to proton beam therapy. However, uncertainties in the proton RBE across the spread-out Bragg peak (SOBP) (typically assumed to be 1.1) may lead to suboptimal treatment plans and unwarranted toxicity to organs-at-risk. Herein, we report a reliable analytical method to determine the proton RBE in vitro along the SOBP and distal fall-off region. The 3D microtissue cassette enables the high throughput assessment of biological assays including clonogenic assay and γ-H2AX assay following a single proton irradiation. Results: Proton RBE values determined using the standard clonogenic assay at 90 %, 50 % and 10 % of cell survival were calculated to be 1.3, 1.5 and 1.6, respectively. This was found to be consistent with the RBE determined using the γ-H2AX for double-strand DNA break repair (1.58 for 10 % cell survival). In addition, we also observed that the high spatial resolution of the cassette can distinguish the minute but significant γ-H2AX foci changes (number, area) in response to small differences in proton radiation dose fraction. Significance: The results validate the reliability of the 3D printed cassette in addressing critical radiobiological issues. This methodology enables high throughput irradiation workflow and consequently reduce the time and resource burden for clinical facilities. This approach could be readily extended to investigate the radiobiological unknown of other emerging radiation therapy modalities based on charged particles.